Lentiviral vectors are increasingly used as efficient gene transfer tools in the experimental and clinical gene therapy treatment of acquired and inherited genetic diseases. Hematopoietic stem cells (HSCs) are characterized by the capacity for self-renewal, as well as multi-lineage differentiation and maintenance of the lymphohematopoietic system throughout life. As such, HSC transplantation (HSCT) has proven to be a powerful therapeutic modality for the treatment of both malignant and nonmalignant disorders. Transduction of lentiviral vectors into HSCs may offer long-term stable expression of a therapeutic gene in both preclinical and clinical settings. The purpose of this chapter is to describe an optimized procedure for lentiviral transduction of mouse HSCs followed by HSCT.